Treatment of vascular degenerative diseases by modulation of endogenous nitric oxide production of activity

ABSTRACT

Atherogenesis and restenosis are treated by long term administration of physiologically acceptable compounds which enhance the level of endogenous nitric oxide in the host. Alternatively, or in combination, other compounds may be administered which provide for short term enhancement of nitric oxide, either directly or by physiological processes. In addition, cells may be genetically engineered to provide a component in the synthetic pathway to nitric oxide, so as drive the process to enhance nitric oxide concentration, particularly in conjunction with the administration of a nitric oxide precursor.

INTRODUCTION

This invention was supported in part by the United States Governmentunder Grant 1KO7HCO266((NHLBI). The U.S. Government may have an interestin this application.

TECHNICAL FIELD

The field of this invention is the treatment of vascular degenerativediseases, particularly atherosclerosis and restenosis.

BACKGROUND

Atherosclerosis and vascular thrombosis are a major cause of morbidityand mortality, leading to coronary artery disease, myocardialinfarction, and stroke. Atherosclerosis begins with an alteration in theendothelium, which lines the blood vessels. The endothelial alterationresults in adherence of monocytes, which penetrate the endotheliallining and take up residence in the subintimal space between theendothelium and the vascular smooth muscle of the blood vessels. Themonocytes absorb increasing amounts of cholesterol (largely in the formof oxidized or modified low-density lipoprotein) to form foam cells.Oxidized low-density lipoprotein (LDL) cholesterol alters theendothelium, and the underlying foam cells distort and eventually mayeven rupture through the endothelium.

Platelets adhere to the area of endothelial disruption and release anumber of growth factors, including platelet derived growth factor(PDGF). PDGF, which is also released by foam cells and alteredendothelial cells, stimulates migration and proliferation of vascularsmooth muscle cells into the lesion. These smooth muscle cells releaseextracellular matrix (collagen and elastin) and the lesion continues toexpand. Macrophages in the lesion elaborate proteases, and the resultingcell damage creates a necrotic core filled with cellular debris andlipid. The lesion is then referred to as a "complex lesion." Rupture ofthis lesion can lead to thrombosis and occlusion of the blood vessel. Inthe case of a coronary artery, rupture of a complex lesion mayprecipitate a myocardial infarction, whereas in the case of a carotidartery, stroke may ensue.

One of the treatments that cardiologists and other interventionalistsemploy to reopen a blood vessel which is narrowed by plaque is balloonangioplasty (approximately 300,000 coronary and 100,000 peripheralangioplasties are performed annually). Although balloon angioplasty issuccessful in a high percentage of the cases in opening the vessel, itunfortunately denudes the endothelium and injures the vessel in theprocess. This damage causes the migration and proliferation of vascularsmooth muscle cells of the blood vessel into the area of injury to forma lesion, known as myointimal hyperplasia or restenosis. This new lesionleads to a recurrence of symptoms within three to six months after theangioplasty in a significant proportion of patients (30-40%).

Because of their great prevalence and serious consequences, it iscritically important to find therapies which can diminish the incidenceof atherosclerosis, vascular thrombosis and restenosis. Ideally, suchtherapies would inhibit the pathological processes associated withatherosclerosis, thereby providing prophylaxis or retarding theprogression of the degenerative process.

As briefly summarized above, these pathological processes are extremelycomplex, involving a variety of different cells which undergo changes intheir character, composition, and activity, as well as in the nature ofthe factors which they secrete and the receptors that are up- ordown-regulated. A substance released by the endothelium, "endotheliumderived relaxing factor" (EDRF), may play an important role ininhibiting these pathologic processes EDRF is now known to be nitricoxide (NO) or a labile nitroso compound which liberates NO. (Forpurposes of the subject invention, unless otherwise indicated, nitricoxide (NO) shall intend nitric oxide or the labile precursor.) Thissubstance has been reported to relax vascular smooth muscle, inhibitplatelet aggregations, inhibit mitogenesis and proliferation of culturedvascular smooth muscle, and leukocyte adherence. NO may have othereffects, either direct or indirect, on the various cells associated withvascular walls and degenerative diseases of the vessel.

Relevant Literature

Girerd, et al. (1990) Circulation Research 67, 1301-1308 report thatintravenous administration of L-arginine potentiatesendothelium-dependent relaxation in the hind limb of cholesterol-fedrabbits. The authors conclude that synthesis of EDRF can be increased byL-arginine in hypercholesterolemia. Rossitch, et al. (1991) J. Clin.Invst. 87, 1295-1299 report that in vitro administration of L-arginineto basilar arteries of hypercholesterolemic rabbits reverses theimpairment of endothelium-dependent vasodilation and reducesvasoconstriction. They conclude that the abnormal vascular responses inhypercholesterolemic animals is due to a reversible reduction inintracellular arginine availability for metabolism to nitric oxide.

Creager, et al. (1992) J. Clin. Invest. 90, 1248-1253, report thatintravenous administration of L-arginine improves endothelium-derivedNO-dependent vasodilation in hypercholesterolemic patients.

Cooke, et al., "Endothelial Dysfunction in Hypercholesterolemia isCorrected by L-arginine," Endothelial Mechanisms of Vasomotor Control,eds. Drexler, Zeiher, Bassenge, and Just; Steinkopff Verlag Darmstadt,1991, pp. 173-181, review the results of the earlier references andsuggest, "If the result of these investigations may be extrapolated,exogenous administration of L-arginine (i.e., in the form of dietarysupplements) might represent a therapeutic adjunct in the treatmentand/or prevention of atherosclerosis."

Cooke, (1990) Current Opinion in Cardiology 5, 637-644 discusses therole of the endothelium in the atherosclerosis and restenosis, and theeffect that these disorders have on endothelial function.

Cooke (1992) J. Clin. Invest. 90, 1168-1172, describe the effect ofchronic administration of oral L-arginine in hypercholesterolemicanimals on atherosclerosis. This is the first demonstration that oralL-arginine supplements can improve the release of NO from the vesselwall. The increase in NO release from the vessel wall was associatedwith a striking reduction in atherosclerosis in hypercholesterolemicanimals. This is the first evidence to support the hypothesis thatincreasing NO production by the vessel wall inhibits the development ofatherosclerosis.

Cooke and Tsao, (1992) Current Opinion in Cardiology 7, 799-804 describethe mechanism of the progression of atherosclerosis and suggest thatinhibition of nitric oxide may disturb vascular homeostasis andcontribute to atherogenesis.

Cooke and Santosa, (1993) In: Steroid Hormones and DysfunctionalBleeding, AAAS Press, review the activities of EDRF in a variety ofroles and suggest that reversibility of endothelial dysfunction may beaffected by the stage of atherosclerosis. They conclude that EDRF is apotent vasodilator, plays a key role in modulating conduit andresistance vessel tone, has important effects on cell growth andinteractions of circulatory blood cells with a vessel wall, and thatdisturbances of EDRF activity may initiate or contribute to septicshock, hypertension, vasospasm, toxemia and atherosclerosis.

Other references which refer to activities attributed to NO or itsprecursor include: Pohl and Busse (1989) Circ. Res. 65:1798-1803;Radomski et al. (1987) Br. J. Pharmacol. 92:181-1187; and Stamler et al.(1989) Circ. Res. 65:789-795; anti-platelet activity); Garg and Hassid(1989) J. Clin. Invest. 83:1774-1777; and Weidinger et al, (1990)Circulation 81:1667-1679; NO activity in relation to vascular smoothmuscle growth); Ross (1986) N. Engl. J. Med. 314:488-500; Bath et al.(1991) Arterioscler. Thromb. 11:254-260; Kubes et al. (1991) Proc. Natl.Acad. Sci. USA 89:6348-6352; Lefer et al. (1990) In: Endothelium-DerivedContracting Factors. Basel, S. Karger, pp. 190-197; NO activity inrelation to leukocyte adhesion and migration); Heistad et al. (1984)Circ. Res. 43:711-718; Rossitch et al. (1991) J. Clin. Invest.87:1295-1299; Yamamoto et al. (1988)ibid 81:1752-1758; Andrews et al.(1987) Nature 327:237-239; Tomita et al. (1990)Circ. Res. 66:18-27;Kugiyama et al. (1990) Nature 344:160-162; Mitchell et al. (1992) J.Vasc. Res. 29:169 (abst.); and Minor et al. (1990) J. Clin. Invest.86:2109-2116; NO activity in relation to hypercholesterolemia); Tanneret al. (1991) Circulation 83:2012-2020; Kuo et al. (1992) Circ. Res.70:f465-476; Drexler et al. (1991) Lancet 338:1546-1550; and Nakanishiet al. (1992) In: Scientific Conference on Functional and StructuralMechanisms of Vascular Control, Snowbird, UT, p. 86 (abstsr.); relationof L-arginine to NO-dependent vasodilation.

SUMMARY OF THE INVENTION

Atherosclerosis and restenosis are treated with agents that enhancenitric oxide formation. The enhancement of endogenous nitric oxideformation inhibits the progression of restenosis and atherosclerosis. Asa prophylaxis or treatment for atherosclerotic susceptible hosts, theagent is chronically administered at an effective dosage. Forrestenosis, the agent may be administered for a limited period sincethis pathological process generally abates 3-6 months after the vascularinjury (i.e. angioplasty or atherectomy). Oral administration ofL-arginine as a dietary supplement will increase NO elaboration andthereby diminish the effects of atherogenesis. Other techniques toenhance NO production may be utilized such as increasing theavailability of NO synthase, for example, as a result of enhancedexpression of NO synthase in the vessel wall, particularly at the lesionsite.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In accordance with the subject invention, common vascular degenerativediseases such as atherosclerosis, vascular thrombosis, and restenosis,are treated prophylactically and/or therapeutically by maintaining anenhanced level of nitric oxide or its precursor in the vessel wall inaccordance with a predetermined regimen over an extended period of time.The enhanced level of nitric oxide (which is intended to include anyprecursor of nitric oxide which results in such enhanced level) can beachieved by modulating the activity, synthesis or concentration of anyof the components associated with the formation of nitric oxide in thenitric oxide synthetic pathway. The enhanced level of nitric oxide maybe a result of administration to the patient of an intermediate in themetabolic pathway to the production of nitric oxide (or itsphysiological equivalent), the enhanced levels of an enzyme associatedwith the production of nitric oxide, or a physiologically acceptableprecursor, which may lead directly or indirectly, to formation of nitricoxide.

One approach is to employ L-arginine as a dietary supplement. This aminoacid may be administered as any physiologically acceptable salt, such asthe hydrochloride salt, glutamate salt, etc. It may also be administeredas a peptide (i.e. poly-L-arginine) so as to increase plasma levels ofthe NO precursor. Naturally occurring sources include protamine. Theadministration of L-arginine or other convenient NO precursor would bein accordance with a predetermined regimen, which would be at least onceweekly and over an extended period of time, generally at least onemonth, more usually at least three months, as a chronic treatment, andcould last for one year or more, including the life of the host. Thedosage administered will depend upon the frequency of theadministration, the blood level desired, other concurrent therapeutictreatments, the severity of the condition, whether the treatment is forprophylaxis or therapy, the age of the patient, the natural level of NOin the patient, and the like. Desirably, the amount of L-arginine orbiologically equivalent compound which is used would generally provide aplasma level in the range of about 0.2 mM to 30 mM. The oraladministration of L-arginine can be achieved by providing L-arginine asa pill, powder, capsule, liquid solution or dispersion, particularlyaqueous, or the like. Various carriers and excipients may find use informulating the NO precursor, such as lactose, terra alba, sucrose,gelatin, aqueous media, physiologically acceptable oils, e.g. peanutoil, and the like. Usually, if daily, the administration of L-argininefor a human host will be about 1 to 12 g per day.

The administration of L-arginine may be administered prophylactically,so as to inhibit atherogenesis or restenosis, or therapeutically afteratherogenesis has been initiated. Thus, for example, a patient who is toundergo balloon angioplasty may have a regimen of L-arginineadministered substantially prior to the balloon angioplasty, preferablyat least about a week or substantially longer. Alternatively, in apatient where atherogenesis is suspected, the administration ofL-arginine may begin at any time. Of particular interest is theincorporation of L-arginine as a supplement in a food, such as a healthbar, e.g. granola, other grains, fruit bars, such as a date bar, figbar, apricot bar, or the like. The amount of L-arginine or theequivalent would be about 2-25 g per dosage or bar, preferably about3-15 g.

Instead of oral administration, intravascular administration may also beemployed, particularly where more rapid enhancement of the nitric oxidelevel in the vascular system is desired (i.e. as with acute thrombosisof a critical vessel), so that combinations of oral and parenteraladministrations may be employed in accordance with the needs of thepatient. Furthermore, parenteral administration may allow for theadministration of compounds which would not readily be transportedacross the mucosa from the gastrointestinal tract into the vascularsystem.

For intravascular administration, a wide variety of individual orcombinations of physiologically acceptable compositions may be employed,which may be provided systemically or in proximity to a lesion site.Thus, one may provide for combinations of peroxy compounds or otheroxygen containing oxidants, where the oxidant is physiologicallyacceptable, in conjunction with a nitrogen source, such as substitutedguanidines, e.g. L-arginine, amidines, or the like. Alternatively, onemay reduce the degradation of endogenous nitric oxide using antioxidants(such as sulfhydryl containing compounds) or compounds that prevent theproduction of oxygen-derived free radicals (such as superoxidedismutase), as it is known that oxygen-derived free radicals (such assuperoxide anion) can inactivate nitric oxide. Thiol compounds may alsofind application, as well as their derivatives, such as disulfides,sulfonic acids, thiol esters, thiono thiol esters, and the like.

Other compounds which may find use include partially oxidized nitrogencompounds, such as hydroxylamines, oxazoles, oxazines, nitrosocompounds, or the like. Physiologically acceptable stable free radicalcompounds, such as nitroxyl compounds, may find use, where the unpairedelectron may be on nitrogen or oxygen, analogous to NO. These compoundswill be, for the most part, synthetic organic compounds generally havinga molecular weight of at least about 100 and usually not more than about2000 D.

Other compositions which may find use include nitrites, includingnitrite esters, e.g. esters of carbonate, thiocarbonate, etc. Thesecompositions may be administered at the site of a lesion to provide forrapid enhancement of nitric oxide concentration, so as to initiate orinhibit the various physiological processes affected by the level ofnitric oxide present and associated with plaque formation or restenosis.Particularly, processes associated with the vascular smooth muscle("VSM") cell proliferation and invasion of the endothelial layer can bemodulated.

Alternatively, one can enhance, either in conjunction with theenhancement of precursors to nitric oxides or independently, componentsof the nitric oxide metabolic pathway. For example, one may enhance theamount of nitric oxide synthetase present in the vessel wall,particularly at the site of lesions. This can be done by localadministration to the lesion site or systemically into the vascularsystem. The synthase may be administered using liposomes, slow releaseparticles, or in the form of a depot, e.g. in collagen, hyaluronic acid,biocompatible gels, vascular stents, or other means, which will providethe desired concentration of the NO-synthase at the lesion site.

Alternatively, cells may be genetically engineered to provide forconstitutive or inducible expression of the synthase. Thus, expressionvectors (viral or plasmid) may be prepared which contain the NO synthasegene and which can be introduced into host cells which will then producehigh concentrations of nitric oxide. These cells may be introduced atthe lesion site or at another site in the host, where the increased NOsynthase activity will maintain an elevated level of NO in the vascularsystem.

Cultured cells can be transfected with expression vectors containing theNO synthase gene ex-vivo and then introduced into the vessel wall, usingvarious intra-arterial or intra-venous catheter delivery systems.Alternatively, techniques of in vivo gene transfer can be employed totransfect vascular cells within the intact vessel in vivo. The NOsynthase gene can be expressed at high constitutive levels or it can belinked to an inducible promoter (which may have tissue specificity) toallow for regulation of NO synthase expression.

DNA constructs are prepared, where a NO synthase gene is joined to anappropriate promoter, either with its native termination region or adifferent termination region, which may provide for enhanced stabilityof the messenger RNA. Constitutive promoters of particular interest willcome from viruses, such as Simian virus, papilloma virus, adenovirus,HIV, Rous sarcoma virus, cytomegalovirus or the like, where thepromoters include promoters for early or late genes, or long terminalrepeats. Endogenous promoters may include the β-actin promoter, orcell-type specific promoters.

A construct is prepared in accordance with conventional techniques, thevarious DNA fragments being introduced into an appropriate plasmid orviral vector, normally a vector capable of replication in a bacterialand/or eucaryotic host. Alternatively, the vector will normally includea marker, which allows for selection of cells carrying the vector, e.g.antibiotic resistance. The vector will normally also include an originwhich is functional in the host for replication. Other functionalelements may also be present in the vector.

Once the vector has been prepared and replicated, it may then be usedfor introduction into host cells. The plasmid vector construct may befurther modified by being joined to viral elements which allow for easeof transfection, may provide a marker for selection, e.g. antibioticresistance, or other functional elements. Introduction of the plasmidvector construct into the host cells may be achieved by calciumphosphate precipitated DNA, transfection, electroporation, fusion,lipofection, viral capsid-mediated transfer, or the like. Alternatively,the NO synthase construct within viral vectors may be introduced bystandard infection techniques. For somatic cell gene therapy, autologouscells will generally be employed, although in some instances allogeneiccells or recombinantly modified cells may be employed. Usually the cellsemployed for genetic modification will be mature endothelial or vascularsmooth muscle cells. Occasionally, the cells employed for geneticmodification will be progenitor cells, particularly early progenitorcells. For example, myoblasts may be employed for muscle cells orhematopoietic stem cells or high proliferative potential cells may beemployed for lymphoid and/or myelomonocytic cells.

Depending upon the nature of the cells, they may be injected into tissueof the same or different cellular nature, they may be injected into thevascular system, where they may remain as mobile cells or home to aparticular site (i.e. the lesion). The number of cells which areadministered will depend upon the nature of the cells, the level ofproduction of the synthase, the desired level of NO synthase in the hostvascular system, at the lesion site, or the like, whether the enhancedlevel of synthase is the only treatment or is used in conjunction withother components of the nitric oxide synthetic pathway, and the like.Therefore, the particular number of cells to be employed will bedetermined empirically in accordance with the requirements of theparticular patient.

These cells may also be introduced into the circulation by first growingthem on the surface of standard vascular graft material (i.e. Dacron orpolytetrafluoroethylene grafts) or other synthetic vascular conduits orvascular bioprostheses.

Alternatively, one may use viral vectors, which are capable of infectingcells in vivo, such as adenovirus or retroviruses. In this case, theviral vector containing the NO synthase gene will be administereddirectly to the site of interest, where it will enter into a number ofcells and become integrated into the cell genome. Thus, one can titerthe desired level of nitric oxide synthase which is secreted, byproviding for one or more administrations of the virus, thusincrementally increasing the amount of synthase which is secreted.Alternatively, one may use modified liposomes as a vehicle forendovascular administration of the vector containing the NO synthasegene. One such modified liposome technique involves mixing the liposomeswith the vector containing NO synthase. Once the NO synthase-containingvector is incorporated into the liposome, the liposomes are coated witha protein (e.g. the viral coat protein of the Hemagglutinating Virus ofJapan) that increases the affinity of the liposome for the vessel wall.

In some situations, the NO synthase gene may be co-transfected with anartificial gene encoding an arginine rich polypeptide susceptible toproteolytic cleavage as an intracellular source of L-arginine. In othersituations, the NO synthase gene may be co-transfected with thesuperoxide dismutase gene, so as to inhibit the degradation of thenitric oxide.

In some situations, acute treatment may be involved, requiring one or afew administrations. This will normally be associated with compoundswhich can act as nitric oxide precursors and are other than naturallyoccurring compounds or are compounds which may be added with naturallyoccurring compounds to enhance the rate of formation of nitric oxide.Thus, one may provide for acute administration of L-arginine andsuperoxide dismutase to increase the nitric oxide concentration over arestricted period of time. These administrations may be independent ofor in conjunction with long term regimens.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1

Anti-atherogenic effects of oral arginine:

Study design: (See, Cooke et al., 1992, supra) Male New Zealand whiterabbits (n=49) were assigned to one of three treatment groups: 10 werefed with normal rabbit chow for ten weeks (Control); 19 received chowenriched with 1% cholesterol (Chol); and 20 received a 1% cholesteroldiet supplemented with 2.5% L-arginine hydrochloride in the drinkingwater (Arg.). Following ten weeks of the dietary intervention, animalswere lightly sedated and the central ear artery cannulated formeasurement of intra-arterial blood pressure, followed by collection ofblood samples for serum chemistries and plasma arginine. Subsequentlythe animals were sacrificed and the left main coronary artery and thethoracic aorta were harvested for studies of vascular reactivity andhistomorphometry. In some animals, blood was collected for studies ofplatelet and monocyte reactivity.

Results: Biochemical and physiological measurements.Hypercholesterolemic animals maintained on oral L-argininesupplementation (Arg) experienced a twofold elevation in plasma argininelevels in comparison to animals on a normal (Control) or 1% cholesterol(Chol) diet alone; the elevation in plasma arginine was maintainedthroughout the course of the study. Serum cholesterol measurements wereelevated equally in both groups receiving the 1% cholesterol diet [50±6vs. 1629±422 vs. 1852±356 mg/dl respectively for Control (=10), Chol(=13), and Arg (=14)]. There were no significant differences inhemodynamic measurements between groups.

Organ chamber studies of isolated vessels: For NO-independent responses,there were no differences between the treatment groups in maximalresponse or sensitivity to norepinephrine (a vasoconstrictor), or tonitroglycerin (a nitrovasodilator). By contrast, NO-dependentrelaxations were attenuated in vessels harvested fromhypercholesterolemic animals with a reduction in the maximal response toacetylcholine, and a reduction in sensitivity to calcium ionophore. Incomparison, vessels harvested from hypercholesterolemic animalsreceiving L-arginine supplementation had improved NO-dependentrelaxation. Similarly, sensitivity of vessels to calcium ionophoreA23187 was greater in the Arg group. In a separate study, the effect ofchronic arginine supplementation to improve NO-dependent relaxation wasconfirmed in the hypercholesterolemic rabbit abdominal aorta.

Histomorphometric studies (planimetry of EVG-stained sections): Ablinded histomorphometric analysis revealed that medial cross-sectionalareas of thoracic aortae were not different between the groups. Bycontrast, the intimal cross-sectional area (i.e. amount ofatherosclerotic plaque) of vessels from hypercholesterolemic animalsreceiving L-arginine supplementation was reduced in comparison to thosefrom animals receiving cholesterol diet alone. In the Arg animals thereduction in the intimal lesion was most pronounced in the ascendingthoracic aorta and left main coronary artery. In the left main coronaryartery of hypercholesterolemic animals receiving arginine, essentiallyno atherosclerotic plaque developed.

Changes in lesion surface area: In a second series of studies, theextent of the thoracic aorta involved by lesions was examined. Inhypercholesterolemic rabbits receiving vehicle (n=6) or L-argininesupplement (n=6), thoracic aortae (from left subclavian artery todiaphragm) were harvested after ten weeks of treatment, bisectedlongitudinally, and stained with oil-red O. Vessels were photographedand vessel and lesion surface area determined by planimetry.Approximately 40% of the total surface area was covered with plaque inthoracic aortae from hypercholesterolemic animals receiving vehicle,whereas in thoracic aortae from arginine-treated hypercholesterolemicanimals, less than 10% of the surface area was covered with plaque.

To summarize, dietary arginine supplementation increases plasma argininelevels, but does not alter serum cholesterol. This is associated withsignificant improvement in NO-dependent vasodilation as judged bybioassay. Finally, the improvement in NO-dependent vasodilation isassociated with reduction in thickness and area of the lesions invessels from hypercholesterolemic animals.

EXAMPLE 2

Inhibition of platelet aggregation by oral L-arginine:

The effect of L-arginine supplementation on platelet reactivity inrabbits that had normal chow (Control; n=6), a 1% cholesterol diet(Chol; n=5), or a 1% cholesterol diet supplemented with oral arginine(Arg; n=6), as detailed above, was examined. Arterial blood obtainedafter central ear artery cannulation was anticoagulated with 13 mMsodium citrate. Platelet-rich suspension was prepared by washingplatelets in calcium-free Krebs-Henseleit solution and resuspending themin Tyrode's solution with albumin. The platelet count was adjusted at2.5×10⁴ platelets/μl by addition of platelet-poor plasma. Aggregationwas initiated by addition of adenosine diphosphate and monitored bystandard nephelometric techniques. In platelets derived from Cholanimals, aggregation was not different in rate or maximum extent incomparison to platelets from Control animals. By contrast, aggregationof platelets from Arg animals was reduced by 50%.

This reduction in platelet aggregation was associated with a twofoldgreater cGMP content in aggregated platelets from arginine-treatedanimals. The reduction of platelet reactivity could be reversed byadministration of N-methylarginine (10⁻⁴ M) in vitro. Therefore, theantiplatelet effect of chronic oral arginine administration can becredited to an increased synthesis of endogenous NO. Furthermore, NOsynthesis may be induced in both the platelets and the endothelium.

EXAMPLE 3

Inhibition of monocyte adherence:

A. Functional Binding Assay: To determine if oral argininesupplementation affects monocyte adherence, blood was collected fromrabbits fed normal chow (=6) a 1% cholesterol diet (=6), or a 1%cholesterol diet supplemented with L-arginine (=6), as described above.Mononuclear cells were purified from blood by Ficoll-paque densitygradient centrifugation. In these preliminary studies, adhesion wasexamined of blood leukocytes to a transformed endothelial cell line,bEnd3 (mouse brain-derived polyoma middle T antigen transformedendothelial cells were examined). The Bend3 cells display the morphologyof endothelial cells, and like human endothelial cells are capable ofuptake of acetylated low-density lipoprotein and express adhesionmolecules in a cytokine-regulable fashion. Cultured cells were grown toconfluence 0.5 cm² Lab-Tek chamber slides (MilesScientific) and treatedwith control medium or with LPS (1 mg/ml) or TNFα (25 U/ml) for 18hours. Cultures were washed with fresh assay buffer, and low, medium, orhigh concentrations of leukocytes (0.75, 1.5, or 3×10⁵ cells/ml,respectively) were added per well. Following a 30-minute incubation on arocking platform at room temperature to allow binding, the slides wereinverted and immersed in buffer containing 2% (v/v) glutaraldehyde, suchthat non-adherent cells were lost and adherent cells were fixed to themonolayer. The adherent mononuclear cells were enumerated usingvideo-light microscopy.

Monocytes from hypercholesterolemic animals (Chol) exhibited greateradherence, consistent with observation by others, that monocytes fromhypercholesterolemic cats or humans exhibit greater adherence tocultured endothelial cells. (deGruijter, et al. (1991) Metabol. Clin.Exp. 40, 1119-1121; Fan, et al. (1991) Virchows Arch. B Cell Pathol. 61,19-27).

In comparison to monocytes derived from vehicle-treatedhypercholesterolemic animals (Chol), those from arginine-treatedhypercholesterolemic animals (Arg) were much more adherent. This datashows that the arginine treatment inhibits adhesion of monocytes to theendothelium, which is the first observable event in atherogenesis.

EXAMPLE 4

Exclusion of the Effect of Enhanced Nitrogen or Caloric Balance asCausing the Observed Results:

To exclude an effect of L-arginine on nitrogen or caloric balance as thecause of these results, six animals received 1% cholesterol dietsupplemented by additional methionine to increase the dietary methioninesix-fold. At ten weeks animals were sacrificed for studies of plateletand vascular reactivity, and histomorphometry. Endothelium-dependentrelaxation, platelet aggregation and intimal thickness were notdifferent than those of animals fed 1% cholesterol diet alone. Theseresults reveal that another amino acid, methionine (which is not aprecursor of NO) does not mimic the effect of the amino acid L-arginine.Therefore lit seems likely that the effect of L-arginine is due to itsmetabolism to nitric oxide, rather than some other effect of amino acidadministration (i.e. change in nitrogen or caloric balance).

EXAMPLE 5

Effect of NO synthase expression on proliferation of vascular smoothmuscle cells:

Cultured rat aortic vascular smooth muscle cells under confluentquiescent conditions were studied. An efficient viral coatprotein-mediated DNA transfer method was employed to transfect the cellswith the NO synthase gene driven by the β-actin promoter and CMVenhancer. This resulted in increased NO synthase activity (as measuredby the arginine-to-citrulline conversion assay) in comparison to controlvector transfected cells. Transfection of the NO synthase genecompletely abolished serum-stimulated DNA synthesis compared to controlvector transfection. These results indicated that increased expressionof NO synthase (associated with increased production of NO) inhibitsexcessive proliferation of vascular smooth muscle cells. This inhibitioncan be correlated with treatment of atherosclerosis and restenosis.

It is evident from the above results, that by enhancing the nitric oxidelevels, by means of nitric oxide precursor compounds or other compoundsin the nitric oxide pathway, substantial benefits will ensue to patientswith vascular degenerative diseases. This treatment will diminish theformation of atherosclerotic plaque and restenosis, by inhibitingadhesion of monocytes and platelets, and by reducing the proliferationof vascular smooth muscle cells.

By virtue of administering to the host, based on a predeterminedregimen, or providing in the host a supply of a component in thesynthetic pathway for production of nitric oxide, so as to maintain amildly elevated level of nitric oxide in the host, particularly at thesite to be treated, the incidence of plaque formation can besubstantially diminished. This can be achieved in a variety of ways: byoral administration in accordance with a predetermined regimen ofvarious compounds associated with nitric oxide formation, e.g.L-arginine; by administration at the site, in a predetermined regimen ofcompounds which can produce nitric oxide, either directly or as a resultof physiologic action of endogenous compounds, e.g. enzymes; byemploying combinations of compounds, which by their action result in theproduction of nitric oxide; or the like. These individualadministrations, can be done independently or in conjunction with aregimen of other compounds associated with the production of nitricoxide.

Alternatively, one may use genetic engineering to introduce a geneassociated with a component in the synthetic pathway for production ofnitric oxide, e.g. nitric oxide synthase, where the enhanced productionof such compounds will have the effect of driving the equilibrium to anenhanced production of nitric oxide. Thus, the subject inventionprovides a plurality of pathways to enhance the synthesis or action ofnitric oxide, or reduce the degradation of nitric oxide, therebyincreasing the effect of endogenous nitric oxide to prevent theformation of vascular lesions and to inhibit restenosis.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method of inhibiting the development ofatherosclerosis or restenosis in the vascular system of a human hostsusceptible to atherosclerosis or restenosis, said methodcomprising:administering to said host in accordance with a predeterminedregimen a member of the group consisting of L-arginine, itsphysiologically acceptable salts and biologically equivalent compoundthereof for enhancement of NO production by NO synthase in an amountsufficient to enhance the level of endogenous NO in the vascular system.2. A method according to claim 1, wherein said biologically equivalentcompound thereof is a peptide comprising L-arginine.
 3. A methodaccording to claim 1, wherein L-arginine is administered.
 4. A methodaccording to claim 2, wherein said L-arginine or physiologicallyacceptable salt is present in a health bar at from about 2-25 g.
 5. Amethod according to claim 1, wherein a physiologically acceptable saltof L-arginine is administered.